Kaposi's sarcoma-associated herpesvirus (KSHV) genomic episomes dynamically move in three- dimensional (3D) nuclear space to ultimately aggregate in reactivating cells. Cellular RNA polymerase II (RNA pol II) also translocates onto the aggregated viral genomes, resulting in the formation of viral transcriptional factories. This previously uncharacterized mechanism represents an additional layer of KSHV gene regulation, which facilitates the reutilization and repurposing of the limited quantities of cellular RNA pol II for effective viral gene expression. Understanding this mechanism may allow us to identify new targets to inhibit viral replication. In this application, we propose a comprehensive study to (1) identify the key cellular proteins involved with viral transcriptional factory structure or function; (2) determine the role of noncoding PAN RNA transcripts and its genomic region in viral transcriptional factory formation; and (3) visualize the physical assembly of viral transcriptional factories in the nuclei of reactivated host cells using live 3D fluorescence microscopy. We will also characterize the molecular architecture of viral transcriptional factories by super-resolution optical microscopy with correlative electron microscopic imaging. Through this multifaceted approach, we hope to understand the role of viral transcriptional factories in the amplification of KSHV gene expression, which may in turn lead to the development of new therapeutic strategies for KSHV-associated malignancies by inhibiting viral replication. In addition, we expect this investigation will shed light on the spatio-temporal (4D) organization of the cell nucleome: using KSHV as a well-defined model chromosome that we can readily track in the host cell nucleus with biochemical and genetic approaches.